Protein phosphorylation is important in the regulation of a wide variety of cellular processes. Regulation of protein activity by phosphorylation of serine, threonine, and tyrosine residues is highly utilized. Histidine, arginine, and lysine residues on proteins are also phosphorylated by cellular processes, but the significance is unknown due to the difficulty of studying these highly unstable modifications. The detection and quantitation of changes in the phosphorylation state of a protein is of great utility in the study of its functional significance.
Standard methods for measuring the state of protein phosphorylation typically involve prelabeling of the added phosphate moiety by incorporation of a radioactive isotope of phosphorous (as a phosphate). Such phosphorylation assays suffer from several methodological pitfalls, including health risks and disposal problems associated with the high amounts of [.sup.32 P]Pi required for the prelabeling experiments, the bother of working with regulated substances, and a lack of site specificity when several sites are phosphorylated in one protein or peptide moiety. As a result of these drawbacks, immuno-chemical based methods for detecting protein phosphorylation state are increasing in popularity. The degree of sensitivity and selectivity achievable with immuno-chemical methodology makes it an attractive alternative (Matsui et al., J. Cell. Bio. 140: 647-657 (1998); Conrad et al., Hybridoma 16: 167-173 (1997)).
Phosphorylation state-dependent monoclonal antibodies specific for a variety of cytoskeletal proteins have been produced and characterized. These antibodies were isolated by immunization protocols in which the specific targeting of phosphorylated epitopes was not the primary objective. More recently, small synthetic phosphopolypeptides have been used to improve the chance of targeting antibody production to epitopes on the phosphorylation sites (Sakaguchi et al., Genes and Dev. 12: 2831-2841 (1998); Matsui et al., J. Cell Bio. 140: 647-657 (1998); Chen et al., FASEB J. 2: A550 (1988); Czernik et al., Methods in Enzymology 201: 264-283 (1991)). While more direct, this method still suffers from the limitation of rapid dephosphorylation of the polypeptide antigen upon immunization which reduces the titer of phospho-specific antibodies. This is particularly a problem when using antigen containing phosphoserine and phosphothreonine, both of which usually are considerably less stable than phosphotyrosine.